Analysis of DNA variations (mutations or polymorphisms) is central to many applications in genetics, genomics as well as in clinical research and diagnostics. One commonly used approach is based on separation of PCR amplified fragment of a target sequence under partial denaturing conditions. The partial denaturation is usually achieved by a chemical denaturant or raising the temperature.
Maintaining an accurate optimum temperature at which mutants in the studied target sequence possesses different conformation from the non-mutated (wildtype) sequence over the duration of electrophoretic or chromatographic separation is generally difficult. A more common approach is to use a temperature gradient during which sooner or later the separated fragments reach their optimum temperature, provided that the temperature range of such gradient is sufficiently wide (Schell et at. 1999). Applying temperature gradients became recently popular in multicapillary electrophoresis systems (Li et al. 2002). With temperature gradients, the reproducibility among different capillaries in an array is better compared to constant temperature control approach. However one problem still remains. The period of time during which the fragment is subjected to its melting optimum depends of the gradient slope i.e. rate at which the temperature is changed. Prior to analysis of an unknown sample it is essential to optimize the temperature settings with respect to the temperature range and gradient slope. Also in some multi-channel systems, it is difficult to control a single-sweep temperature gradient profile reproducibly in all channels at once. As a result the resolution as well as channel-to-channel reproducibility is often not optimal. Finally the overall sample throughput of a single-sweep gradient system is limited due to the relatively long time duration required for the gradient to be completed over the entire course of the separation.
It is an object of this invention to provide a method for separation of compounds (such as DNA molecules) under partially or fully denaturing conditions.
It is a further object of the invention to provide a method not requiring complex optimization of separation parameters such as the denaturant gradient slope.
It is a further object of the invention to provide a method applicable to detection of DNA mutations and polymorphisms using multiple-injection technology for increased sample throughput.
It is a further object of the invention to provide a method applicable to multi-channel separation instruments without a need of complex changes in instrumentation design.